Abstract
Only two polyethylene glycol terephthalate (PET)-degrading enzymes have been reported, and their mechanism for the biochemical degradation of PET remains unclear. To identify a novel PET-degrading enzyme, a putative cutinase gene (cut190) was cloned from the thermophile Saccharomonospora viridis AHK190 and expressed in Escherichia coli Rosetta-gami B (DE3). Mutational analysis indicated that substitution of Ser226 with Pro and Arg228 with Ser yielded the highest activity and thermostability. The Ca2+ ion enhanced the enzyme activity and thermostability of the wild-type and mutant Cut190. Circular dichroism suggested that the Ca2+ changes the tertiary structure of the Cut190 (S226P/R228S), which has optimal activity at 65–75 °C and pH 6.5–8.0 in the presence of 20 % glycerol. The enzyme was stable over a pH range of 5–9 and at temperatures up to 65 °C for 24 h with 40 % activity remaining after incubation for 1 h at 70 °C. The Cut190 (S226P/R228S) efficiently hydrolyzed various aliphatic and aliphatic-co-aromatic polyester films. Furthermore, the enzyme degraded the PET film above 60 °C. Therefore, Cut190 is the novel-reported PET-degrading enzyme with the potential for industrial applications in polyester degradation, monomer recycling, and PET surface modification. Thus, the Cut190 will be a useful tool to elucidate the molecular mechanisms of the PET degradation, Ca2+ activation, and stabilization.
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Acknowledgments
This work was financially supported by funding for F. K. from the Institute for Fermentation, Osaka (Japan). We thank Mr. M. Ono (Showa Denko K. K.) for kindly providing agricultural PBSA and PBS films. We acknowledge Mr. Y. Iijima, Mr. T. Matsunami, Mr. T. Takeda, and Ms. M. Nakajima for their technical assistance during this work. Dr. T. Kawabata (Inst. Protein Res., Osaka Univ.) was helpful in the structural analysis of Cut190 via 3D modeling. We are grateful to Dr. A. Sugiyama (Research Institute for Sustainable Humanosphere, Kyoto Univ.) for the LC-IT-TOF-MS analysis. We are also grateful to Dr. H. Nakajima, Center for Fiber and Textile Science, Kyoto Institute of Technology, for his valuable discussion and advice with regard to the whole polymer properties. American Journal Experts (www.aje.com) edited the manuscript.
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Kawai, F., Oda, M., Tamashiro, T. et al. A novel Ca2+-activated, thermostabilized polyesterase capable of hydrolyzing polyethylene terephthalate from Saccharomonospora viridis AHK190. Appl Microbiol Biotechnol 98, 10053–10064 (2014). https://doi.org/10.1007/s00253-014-5860-y
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DOI: https://doi.org/10.1007/s00253-014-5860-y